ABSTRACT The mitochondrial apoptotic pathway plays a critical role in the response to various cellular stresses, including targeted anticancer therapies. This pathway is regulated by interactions between various members of the BCL2 family of proteins. In particular, BAX and BAK play an indispensible role in this pathway by permeabilizing the mitochondrial outer membrane (MOM). While BAX plays a predominant role in epithelial tissues, especially in postnatal life, BAK is particularly abundant in normal white blood cells, leukemia cell lines, and clinical leukemia specimens. Our previous studies have demonstrated that BAK activation is initiated by two distinct processes: i) Transient binding of BH3-only members of the BCL2 family in response to certain stimuli (e.g., transient binding of NOXA, which is upregulated in response to the NEDD8 activating enzyme inhibitor pevonedistat), and ii) concentration-dependent BAK autoactivation, a process we initially described. Once activated, BAK forms multimers that permeabilize the MOM. Our recent studies indicate that this MOM permeabilization involves the action of a C-terminal lipid binding domain that is externalized upon BAK activation and interacts with the MOM lipid cardiolipin. Counterbalancing this pro-apoptotic effect, however, BAK can be bound and neutralized by anti- apoptotic BCL2 paralogs in lymphohematopoietic cell lines and primary acute myeloid leukemia (AML) specimens. Importantly, the response of these cells to BH3 mimetics, proapoptotic small molecules that selectively bind and neutralize BCL2, BCLXL and/or MCL1, reflects which of the anti-apoptotic BCL2 family member(s) constitutively bind BAK. Collectively, these observations lead to the hypothesis that AMLs with higher BAK levels will harbor more constitutively activated BAK and will be particularly sensitive to BH3 mimetics as well as targeted therapies that activate BH3-only proteins. We now propose three aims that will test this hypothesis and provide additional insight into the action of BAK in AML during anti-leukemic therapy. First, we will assess the mechanisms responsible for high BAK expression in some AMLs but not others because high BAK expression contributes to BAK autoactivation. Second, we will determine the biochemical basis for BAK autoactivation and subsequent restraint by anti-apoptotic BCL2 family members because this partially- activated-and-then-restrained BAK is the species poised to kill leukemia cells upon exposure to BH3 mimetics and targeted therapies that upregulate BH3-only proteins. Third, we will assess the relationship between high BAK expression, BAK restraint by various anti-apoptotic BCL2 family members, and response of clinical AML to a novel pevonedistat-containing combination undergoing early phase clinical testing, thereby assessing the potential importance of constitutive BAK activation in the clinical setting. These studies, which build on our recent advances in understanding the action of BAK at the molecular level, are collectively designed to enhance current understanding of BCL2 family biology and simultaneously provide new insight into a potentially important determinant of AML sensitivity in the clinic.